p11 Expression in Human Bronchial Epithelial Cells Is Increased by Nitric Oxide in a cGMP-dependent Pathway Involving Protein Kinase G Activation

• Published in: The Journal of biological chemistry 2001-11, Vol.276 (48), p.44613-44621
• Main Authors: Pawliczak, R, Cowan, M J, Huang, X, Nanavaty, U B, Alsaaty, S, Logun, C, Shelhamer, J H
• Format: Article
• Language: English
• Subjects: Alkaloids - pharmacology; Annexin A2; Bronchi - metabolism; Calcium-Binding Proteins - biosynthesis; Calcium-Binding Proteins - metabolism; Carbazoles; Cell Nucleus - metabolism; Chloramphenicol O-Acetyltransferase - metabolism; Cyclic GMP - analogs & derivatives; Cyclic GMP - metabolism; Cyclic GMP - pharmacology; Cyclic GMP-Dependent Protein Kinases - antagonists & inhibitors; Cyclic GMP-Dependent Protein Kinases - metabolism; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors - pharmacology; Epithelial Cells - metabolism; Genes, Dominant; Genes, jun - genetics; Genes, Reporter; Immunoblotting; Indoles; Mutation; Nitric Oxide - metabolism; Nitrogen Oxides; Plasmids - metabolism; Promoter Regions, Genetic; Protein Binding; RNA, Messenger - metabolism; S100 Proteins; Spermine - analogs & derivatives; Spermine - pharmacology; Thionucleotides - pharmacology; Time Factors; Transcription Factor AP-1 - metabolism; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M104993200

The effect of nitric oxide on p11 expression was studied in an immortalized human bronchial epithelial cell line (BEAS-2B cells). Three nitric oxide donors were used: spermine NONOate (SP), (±)- S -nitroso- N -acetylpenicillamine (SNAP), and S -nitrosoglutathione (SNOG). All three nitric oxide donors had similar effects resulting in dose-dependent and time-dependent accumulation of p11 protein and an increase of steady-state p11 mRNA. Studies using a reporter gene containing the region from −1499 to +89 of the p11 promoter demonstrated an increase in transcriptional activity after stimulation with NO donors for 4 h. These effects were abolished at the promoter and protein level using protein kinase G inhibitors (KT5823 and R p -8-pCPT-cGMPS). Incubation of transfected cells with a cell permeable cGMP analogue (8-Br-cGMP) resulted in a dose-related increase of promoter activity. An electrophoretic mobility shift assay of nuclear proteins extracted from BEAS-2B cells identified an AP-1 site located at −82 to −77 of the p11 promoter region as an NO- and cGMP- dependent response element. These data were confirmed using a c -jun dominant negative mutant vector and a c -jun expression plasmid. Therefore, we conclude that nitric oxide-induced p11 expression in human bronchial epithelial cells is mediated at least in part through increased binding of activator protein one to the p11 promoter.